PROJECT SUMMARY Myeloproliferative neoplasms (MPNs) are a group of bone marrow diseases that show excessive myeloid cell production with an increased risk of developing thrombosis and evolving to acute myeloid leukemia. V617F driver mutation of JAK2 is one of the leading causes of MPNs. The discovery of this mutation led to the development of JAK inhibitors to treat MPNs. However, JAK inhibitors are not curative. In addition, MPN patients treated with JAK inhibitor often develop drug resistance and severe side effects due to the indispensable roles of JAK2 in normal hematopoiesis. We have been studying new approaches to treat MPNs, especially focusing on the downstream effectors of the JAK2 pathway. Our recently published studies funded by the current R01 revealed that loss of Pleckstrin-2 (Plek2), a novel target of the JAK2-STAT5 pathway, ameliorated JAK2V617F-induced myeloproliferative phenotypes, and more importantly reverted vascular occlusions and lethality of the JAK2V617F MPN mouse model. Given the significance of Plek2 in MPN pathogenesis, we have identified lead compounds of Plek2 small molecule inhibitors using in silico-based high- throughput screenings and cell-based assays. Our novel unpublished preliminary data further reveal that Plek2 binds to several PI3K effectors and loss of Plek2 reduces Akt activation. Preliminary in vivo evidence also demonstrates that Plek2 is critical for the PI3K-Akt pathway in that Plek2 knockout ameliorated myeloproliferation and significantly extended the survival of Pten hematopoietic specific knockout mice. These findings lead us to hypothesize that Plek2 functions as a central hub to connect the JAK2-STAT and the PI3K- Akt pathways and promote myeloproliferation. To test this hypothesis and investigate the mechanism of function of Plek2, we will use 1) in vitro biochemical and molecular studies to determine how Plek2 enhances the PI3K-Akt signaling, 2) Pten hematopoietic specific knockout mouse model to reveal the functions of Plek2 in vivo, and 3) pre-clinical MPN models and MPN patient samples to establish the efficacy of Plek2 inhibitors. Successful completion of this project will lead to novel insights into the role of Plek2 in the pathogenesis of MPNs and lay the foundation for clinical trials using Plek2 inhibitors as single agents or in combination with other compounds to treat MPNs.